Delayed replication of human cytomegalovirus (CMV) was initiated in human embryonic fibroblasts using partially ultraviolet light-inactivated virus stock. Cellular [high molecular weight (HMW)] DNA extracted from CMV-infected cell cultures demonstrated a substantial increase in transforming activity after introduction into hamster embryo fibroblasts relative to HMW DNA extracted from mock-infected cells. The transforming activity of HMW DNA varied between 0.01 and 0.25 foci/micrograms DNA. HMW DNA isolated from CMV-infected cells after initiation of viral DNA synthesis demonstrated a significant decrease in the induction of morphologically transformed foci. The transforming activity of HMW DNA was unaffected by HindIII or XbaI endonuclease digestion, but it was sensitive to sonication and EcoRI endonuclease and DNase treatment. Six cell lines were established from the foci of morphologically altered cells. Cells of these lines demonstrated loss of contact inhibition, replication in semisolid agarose or in medium containing low serum, a high saturation density, and tumorigenicity when implanted into hamsters. Histopathological examination of the tumors identified the tissues as fibrosarcomas. In situ hybridization of cells isolated from foci of morphologically altered cells or Southern blot analysis of DNA isolated from either the cell lines or tumors did not demonstrate the presence of sequences with homology to viral DNA using the transforming region or the entire viral DNA as a probe. The lack of viral DNA sequences as well as the similar phenotypic characteristics of cell lines and tumors suggest that alteration(s) induced by CMV may occur in specific region(s) of cellular DNA.